Cerebrospinal fluid-contacting neurons in the paraventricular organ and in the spinal cord of the quail embryo: a fluorescence-histochemical study

Although the cerebrospinal fluid-contacting neurons of the avian paraventricular organ exhibit considerable amounts of catecholamines, they show no tyrosine hydroxylase immunoreactivity. In the quail embryo, the development of these neurons has been studied using the paraformaldeyde-glutaraldeyde method for the fluorescence-histochemical localization of catecholamines. The timing of the appearance of catecholamine fluorescence in cerebrospinal fluid-contacting neurons and that in catecholamine-containing neurons of the brainstem have been compared. The first neurons displaying catecholamine fluorescence are found within the locus coeruleus and the nucleus subcoeruleus ventralis on the 5.5th day of incubation. Catecholaminergic neuronal groups of the medulla and mesencephalon can be identified by embryonic day 7, and fluorescent cerebrospinal fluid-contacting neurons of the hypothalamic paraventricular organ can be first recognized at the 8th day of incubation. If the catecholamine content of cerebrospinal fluid-contacting neurons that lack tyrosine hydroxylase depends upon an uptake mechanism, it may be significant that, in fluorescence-histochemical preparations, these neurons can be identified 1–3 days later than those in which catecholamines are synthesized and from which catecholamines are released at an earlier developmental stage. Moreover, cerebrospinal fluid-contacting neurons that have previously been shown to be tyrosine-hydroxylase immunoreactive, and that lie at the spinal-medullary junction display a different developmental pattern. By fluorescence histochemistry, they can be detected only by embryonic day 10.5. The chemical, developmental and topographical differences suggest that the catecholamine-containing cerebrospinal fluid-contacting elements of the paraventricular organ and those of the spinal cord represent two different subsets of cerebrospinal fluid-contacting neurons whose respective functional roles remain to be investigated.     

An attempt to record neuronal activity in the paraventricular organ of Rana esculenta by means of a direct access to the infundibular recess

Summary In submammalian vertebrates, the paraventricular organ (PVO) of the third ventricle is a complex circumventricular structure composed of cerebrospinal fluid-contacting neurons and corresponding deeper formations of nerve cells. A new in-vivo technique enables us to approach the paraventricular organ of the frog, Rana esculenta, via the lobus infundibularis. In this preparation, blood flow in the capillary loops beneath the PVO and the flow of the cerebrospinal fluid in the infundibular recess can be directly observed. Electrical recordings of neural activity in and near the PVO show continuous and phasic, spontaneous activity. Light stimulation of the retina and direct illumination of the brain were not followed by alterations of nerve cell activity. A major problem in the electrophysiological investigation is the diminution in spontaneous activity of the recorded neurons after exchange of CSF.     

Stage-dependency of apoptosis and the blood-testis barrier in the dogfish shark (Squalus acanthias): cadmium-induced changes as assessed by vital fluorescence techniques

Naturally occurring heavy metals and synthetic compounds are potentially harmful for testicular function but evidence linking heavy metal exposure to reduced semen parameters is inconclusive. Elucidation of the exact stage at which the toxicant interferes with spermatogenesis is difficult because the various germ cell stages may have different sensitivities to any given toxicant, germ cell development is influenced by supporting testicular somatic cells and the presence of inter-Sertoli cell tight junctions create a blood-testis barrier, sequestering meiotic and postmeiotic germ cells in a special microenvironment. Sharks such as Squalus acanthias provide a suitable model for studying aspects of vertebrate spermatogenosis because of their unique features: spermatogenesis takes place within spermatocysts and relies mainly on Sertoli cells for somatic cell support; spermatocysts are linearly arranged in a maturational order across the diameter of the elongated testis; spermatocysts containing germ cells at different stages of development are topographically separated, resulting in visible zonation in testicular cross sections. We have used the vital dye acridine orange and a novel fluorescence staining technique to study this model to determine (1) the efficacy of these methods in assays of apoptosis and blood-testis barrier function, (2) the sensitivity of the various spermatogonial generations in Squalus to cadmium (as an illustrative spermatotoxicant) and (3) the way that cadmium might affect more mature spermatogenic stages and other physiological processes in the testis. Our results show that cadmium targets early spermatogenic stages, where it specifically activates a cell death program in susceptible (mature) spermatogonial clones, and negatively affects blood-testis barrier function. Since other parameters are relatively unaffected by cadmium, the effects of this toxicant on apoptosis are presumably process-specific and not attributable to general toxicity.This study was mainly carried out during summer fellowships at the Mount Desert Island Biological Laboratory, Salsbury Cove, Maine, USA, and partly with financial support from the National Research Foundation of South Africa.     

Vasoactive intestinal peptide-immunoreactive cerebrospinal fluid-contacting neurons in the reptilian lateral septum nucleus accumbens

By means of immunocytochemical demonstration of vasoactive intestinal peptide (VIP) an accumulation of cerebrospinal fluid (CSF)-contacting neurons was found in a circumscribed region of the nucleus accumbens/lateral septum of eleven reptilian (chelonian, lacertilian, ophidian, crocodilian) species. Basal processes of these cells contribute to a subependymal plexus whose density displays considerable interspecific variation. VIP-immunoreactive nerve fibers occur also in the lateral septum and the nucleus accumbens where they encompass immunonegative cells in a basket-like pattern. The CSF-contacting neurons are surrounded by columnar ependymocytes frequently arranged in a pseudostratified manner. These specialized arrays of ependymal cells, however, occupy a more extended area than the VIP-immunoreactive CSF-contacting neurons and can be traced from the rostro-ventral pole of the lateral ventricle to the interventricular foramen. These observations suggest the existence of a telencephalic site of CSF-contacting neurons which may be more widespread than hitherto thought and which may participate in a circumventricular system of the lateral ventricle. Previous studies mainly performed with birds indicate that the VIP-immunoreactive CSF-contacting neurons of the nucleus accumbens might form a part of the encephalic (extraretinal and extrapineal) photoreceptor. However, further experiments are required to test this supposition since the VIP-immunoreactive neurons of the nucleus accumbens remained unlabeled by antibodies against bovine rodopsin and chicken cone-opsin in all eleven species analysed in this investigation.     

Regulatory peptides in the pancreas of two species of elasmobranchs and in the Brockmann bodies of four teleost species

Summary Immunohistochemistry was used to localize regulatory peptides in endocrine cells and nerve fibres in the pancreas of two species of elasmobranchs (starry ray,Raja radiata and spiny dogfish,Squalus acanthias), and in the Brockmann bodies of four teleost species (goldfish,Carassius auratus, brown troutSalmo trutta, rainbow trout,Oncorhynchus mykiss and cod,Gadus morhua). In the elasmobranchs, the classical pancreatic hormones somatostatin, glucagon and insulin were present in endocrine cells of the islets. In addition, endocrine cells were labelled with antisera to enkephalins, FMRF-amide, gastrin/cholecystokinin-(CCK)/caerulein, neurotensin, neuropeptide Y (NPY), and peptide YY (PYY). Nerve fibres were demonstrated with antisera against bombesin, galanin and vasoactive intestinal polypeptide (VIP). These nerve fibres innervated the walls of blood vessels, in the exocrine as well as the endocrine tissue. In the four teleost species immunoreactivity to somatostatin, insulin and glucagon was intense in the Brockmann bodies. Cells were labelled with antisera to enkephalin, neurotensin, FMRFamide, gastrin/CCK/ caerulein, NPY, PYY and VIP. Only a few nerve fibres were found with antisera against dopamine--hydroxylase (DBH, cod), enkephalin (met-enkephalin-Arg-Phe, cod), bombesin (cod), gastrin/CCK/caerulein (cod) and VIP. Galanin-like-immunoreactive fibres were numerous in the Brockmann bodies of all teleosts examined. Immunoreactivity to calcitonin gene-related peptide (CGRP), substance P, tyrosine hydroxylase (TH), and phenyl-N-methyl transferase (PNMT) could not be found in any of the species studied.     

Immunohistochemical localization of serotonin,leu-enkephalin,tyrosine hydroxylase,and substance P within the visceral sensory area of cartilaginous fish

Summary We examined the distribution of immunoreactivity to serotonin (5-HT), leu-enkephalin (LENK), tyrosine-hydroxylase (TH), and substance P (SP) within the primary visceral sensory region of cartilaginous fish. Two genera of sharks, Squalus and Heterodontus, a skate, Raja, a ray, Myliobatis, and a holocephalian, Hydrolagus, were used. Cranial nerves, VII, IX, and X enter the visceral sensory complex from the lateral aspect and divide it into lobes. Based on sagittally cut sections, there are four lobes in Hydrolagus and five in Squalus, corresponding to the number of gill arches. The neurochemicals are differentially distributed within each lobe. LENK+ and 5-HT+fibers are located in all regions within the visceral sensory complex. SP+fibers are extremely dense in a dorsolateral subdivision and do not extend as far ventrally as 5-HT+ and LENK+fibers. The lobes lack 5-HT+cells, but contain a few LENK+ and SP+cells. Many TH+cells are distributed in dorsomedial portions of the complex, but there are few TH+fibers. Thus, the visceral sensory area of cartilaginous fish contains several divisions that can be distinguished by their neurochemical content.     

Immunocytochemical and electron-microscopic study of the elasmobranch nucleus sacci vasculosi

Summary The elasmobranch nucleus sacci vasculosi was studied by means of electron microscopy (in the dogfish) and immunocytochemistry (in the dogfish and the skate) by using antibodies against tyrosine hydroxylase, alpha-melanocyte-stimulating hormone, somatostatin, serotonin, and substance P. Ultrastructural study of the dogfish nucleus sacci vasculosi shows the presence of medium-sized cells that possess numerous mitochondria but that have no dense-core vesicles in the cytoplasm or in cell processes. Fibres of the conspicuous tractus sacci vasculosi have a beaded appearance and form conventional synapses with dendrites and cell perikarya of the nucleus sacci vasculosi. The perikarya of this hypothalamic nucleus were not immunoreactive to any of the antibodies tested, and fibres immunopositive to tyrosine hydroxylase, alpha-melanocyte-stimulating hormone, somatostatin, serotonin, and substance P were scarce within this nucleus, in both the dogfish and the skate. Dorsal to the nucleus sacci vasculosi, there are numerous positive neuronal processes in addition to many small neurons that show immunoreactivity to alpha-melanocyte-stimulating hormone, somatostatin and tyrosine hydroxylase. Two types of neuron occur in this dorsal region, displaying dense-core vesicles of either 100–160 nm or 60–100 nm diameter in their cytoplasm; they were identified as peptide-containing and monoamine-containing neurons, respectively. The neuropil of this region has a significantly different ultrastructure from that of the nucleus sacci vasculosi, with many processes containing dense-core vesicles. This group of neurons, located dorsal to the nucleus sacci vasculosi and showing (a) immunoreactivity to neuropeptides or to monoamine-synthesizing enzyme, and (b) cytoplasm with dense-core vesicles, was considered not to be a part of the nucleus sacci vasculosi but rather part of the nucleus tuberculi posterioris. These results support the non-peptidergic and non-aminergic character of the nucleus sacci vasculosi.     

Immunocytochemical identification of photoreceptor proteins in hypothalamic cerebrospinal fluid-contacting neurons of the larval lamprey (Petromyzon marinus)

Antibodies directed against different visual pigment opsins, and an antibody raised against the C terminal of the -subunit of retinal G protein (transducin) labelled cerebrospinal fluid-contacting cells located within the hypothalamus (postoptic commissural nucleus and ventral hypothalamic nucleus) of ammocoete lampreys (Petromyzon marinus). These antibodies also labelled photoreceptor cells within the retina and the pineal and parapineal organs, but no other areas of the brain. Despite considerable behavioural and physiological evidence for the existence of deep brain photoreceptors, numerous studies have failed to identify photoreceptor proteins within the basal brain. The results presented in this paper support our recent results in the lizard Anolis carolinensis, suggesting that a group of cerebrospinal fluid-contacting neurons within the vertebrate brain have a photosensory capacity. We speculate that these cells mediate extraocular and extrapineal photoreception in nonmammalian vertebrates.     

Granulated peripolar epithelial cells in the renal corpuscle of marine elasmobranch fish

Summary Granulated epithelial cells at the vascular pole of the renal corpuscle, peripolar cells, have been found in the kidneys of five species of elasmobranchs, the little skate (Raja erinaced), the smooth dogfish shark (Mustelus canis), the Atlantic sharpnose shark (Rhizoprionodon terraenovae), the scalloped hammerhead shark (Sphryna lewini), and the cow-nosed ray (Rhinoptera bonasus). In a sixth elasmobranch, the spiny dogfish shark (Squalus acanthias), the peripolar cells could not be identified among numerous other granulated epithelial cells. The peripolar cells are located at the transition between the parietal epithelium of Bowman's capsule and the visceral epithelium (podocytes) of the glomerulus, thus forming a cuff-like arrangement surrounding the hilar vessels of the renal corpuscle. These cells may have granules and/or vacuoles. Electron microscopy shows that the granules are membrane-bounded, and contain either a homogeneous material or a paracrystalline structure with a repeating period of about 18 nm. The vacuoles are electron lucent or may contain remnants of a granule. These epithelial cells lie close to the granulated cells of the glomerular afferent arteriole. They correspond to the granular peripolar cells of the mammalian, avian and amphibian kidney. The present study is the first reported occurrence of peripolar cells in a marine organism or in either bony or cartilagenous fish.     

Vasoactive intestinal polypeptide (VIP)-containing neurons: distribution throughout the brain of the chick (Gallus domesticus) with focus upon the lateral septal organ

The distribution of VIP-like perikarya and fibers was determined throughout the chick brain. The most rostral immunoreactive perikarya were found to be cerebrospinal fluid-contacting neurons in the pars medialis of the lateral septal organ. Additional data were presented supporting the idea that the lateral septal organ is another circumventricular organ within the brain of birds (Kuenzel and van Tienhoven 1982). A large group of immunoreactive perikarya was found in the lateral hypothalamic area and appeared continuous with immunoreactive neurons in the anterior medial and ventromedial hypothalamic nuclei (n). A few perikarya were located in the paraventricular hypothalamic n. A number of immunoreactive neurons were found within and about the infundibular and inferior hypothalamic n., none however was immunoreactive cerebrospinal fluid-contacting neurons. Immunoreactive perikarya were found predominantly in laminae 10–11 of the stratum griseum et fibrosum superficiale. A few scattered perikarya were found ventromedial to the n. tegmenti pedunculo-pontinus pars compacta and locus ceruleus. Some of the immunoreactivity was unusual, being very homogeneous within the cell body with little evidence of the material in the axon or dendrites. Perikarya were found in the central gray, n. intercollicularis, and area ventralis of Tsai. The most caudal structure showing immunoreactive neurons was the n. reticularis paragigantocellularis lateralis. Brain areas containing the most abundant immunoreactive fibers, listed from the rostral-most location, were found in the ventromedial region of the lobus parolfactorius and the lateral septal n. Continuing caudally, there were immunoreactive fibers within the periventricular hypothalamic n.; some of the fibers were found to travel for some distance parallel to the third ventricle. Dense immunoreactive fibers were found in the tractus cortico-habenularis et cortico-septalis, medial habenular n. and posterior and dorsal n. of the archistriatum. A number of areas had what appeared to be baskets of immunoreactive fibers (perhaps immunoreactive terminals) surrounding non-reactive perikarya. Brain areas containing terminals included the piriform cortex, area ventralis of Tsai, interpeduncular n., and specific regions of the stratum griseum et fibrosum superficiale. A very dense immunoreactivity occurred within the external zone of the median eminence, the dorsolateral parabrachial n., and n. tractus solitarii. Vasoactive intestinal polypeptide appears to be a useful peptide for defining the neuroanatomical constituents of the visceral forebrain in birds.     

C-PON immunoreactive neurons in the neostriatum of the hedgehog (Erinaceus europaeus): a correlated light- and electron-microscopic study

The present study provides light- and electronmicroscopic immunocytochemical data on the presence of neurons that are immunoreactive to the C-terminal flanking peptide of neuropeptide Y, C-PON, in the neostriatum of the hedgehog (Erinaceus europaeus). Positive neurons have mostly fusiform or round perikarya from which two to four poorly branched processes arise. Immunostained fibers and puncta are also evenly distributed throughout the neostriatum. Ultrastructurally, each neuron exhibits a deeply invaginated nucleus surrounded by abundant cytoplasm with a well-developed rought endoplasmic reticulum and Golgi apparatus. Positive neurons receive symmetric and asymmetric synapses from unlabeled terminals. The results of this study can be correlated with previous findings, as the C-PON-positive neurons of the hedgehog resemble medium-sized neostriatal neurons that are known to be local circuit neurons exhibiting C-PON in the rat. Thus, a high degree of C-PON neuronal system phylogenetic conservation and function can be postulated for the neostriatum of mammals.     

Distribution of FMRFamide-like immunoreactive neurons in the central nervous system of the snail Helix pomatia

Summary The distribution of FMRFamide-like immunoreactive (FLI) neurons and their morphological characteristics have been investigated in the central nervous system of the snail, Helix pomatia L. Approximately phageal ganglion complex. More than 50% of the FLI neurons were located in the cerebral ganglia. The FLI neurons could be divided into four groups according to size: (i) giant neurons (over 100 m); (ii) large neurons (80–100 m); (iii) medium-sized neurons (40–70 m); (iv) small neurons (12–30 m). They were distributed i) in groups or clusters, typical of small neurons and ii) in solitary form or in groups comprising 2–3 cells, typical of large and giant neurons. Giant and large neurons revealed only limited arborizations in the neuropil, but rich branching towards and in the peripheral nerves. Some of the small neurons had extensive arborizations of varicose fibers in the neuropil. They may therefore play some role in integratory processes. Varicose FLI fibers were visualized in the cell body layer of the different ganglia, and in the neural sheath of both the ganglia and the peripheral nerves. We propose a multifunctional involvement of FLI neurons and FMRFamide-like neuropeptides in the Helix nervous system: (i) a synaptic or modulatory role in axo-axonic interactions in the neuropil; (ii) a direct influence on neuronal cell bodies in the cortical layer, (iii) innervation of different peripheral organs; and (iv) remote neurohormonal control of peripheral events through the neural sheath.     

Immunocytochemical localization of S-100 protein in the hypophysis and saccus vasculosus of the elasmobranchs Mustelus manazo and Scyliorhinus torazame

Summary S-100 protein-immunoreactive cells were demonstrated by immunocytochemical procedures in the hypophysis and saccus vasculosus of two species of elasmobranchs (Mustelus manazo and Scyliorhinus torazame). In the saccus vasculosus of M. manazo, immunoreactivity was detectable exclusively in the fibrous portions interposed between the epithelial layer and the blood vessels. In the neurohypophysis, tanycytes and astrocytes of the median eminence were immunostained, but only a few labeled cells were found in the neurointermediate lobe. In S. torazame, the neurohypophysis displayed a similar distribution of immunoreactivity, but there were no labeled cells in the saccus vasculosus. In both species, none of the glandular cells of the hypophysis displayed immunoreactivity. Electron-microscopic examination showed that the immunostained cells in the saccus vasculosus correspond to astrocytes.     

Distribution and anatomy of GABA-like immunoreactive neurons in the central and peripheral nervous system of the snail Helix pomatia

Gamma-aminobutyric acid (GABA)-like immunoreactive neurons were studied in the central and peripheral nervous system of Helix pomatia by applying immunocytochemistry on whole-mount preparations and serial paraffin sections. GABA-immunoreactive cell bodies were found in the buccal, cerebral and pedal ganglia, but only GABA-immunoreactive fibers were found in the viscero-parietal-pleural ganglion complex. The majority of GABA-immunoreactive cell bodies were located in the pedal ganglia but a few could be found in the buccal ganglia. Varicose GABA-ir fibers could be seen in the neuropil areas and in distinct areas of the cell body layer of the ganglia. The majority of GABA-ir axonal processes run into the connectives and commissures of the ganglia, indicating an important central integrative role of GABA-immunoreactive neurons. GABA may also have a peripheral role, since GABA-immunoreactive fibers could be demonstrated in peripheral nerves and the lips. Glutamate injection did not change the number or distribution of GABA-immunoreactive neurons, but induced GABA immunoreactivity in elements of the connective tissue ensheathing the muscle cells and fibers of the buccal musculature. This shows that GABA may be present in different non-neural tissues as a product of general metabolic pathways.     

Distribution of catch-relaxing peptide (CARP)-like immunoreactive neurons in the central and peripheral nervous system of Helix pomatia

Immunocytochemistry was performed on the nervous system of Helix by the use of an antibody raised against a myotropic neuropeptide, the catch-relaxing peptide (CARP), isolated from Mytilus edulis. In each ganglion of the central nervous system of Helix pomatia, numerous CARP-immunoreactive cell bodies and a dense immunoreactive fiber system could be observed with a dominancy in the cerebral and pedal ganglia. The majority of the immunoreactive neurons are unipolar, although multipolar neurons also occur. In the neuropil areas, CARP-immunoreactive fibers show extensive arborization, which may indicate a central role of CARP. CARP-immunoreactive elements could be observed in each investigated peripheral nerve and peripheral areas, namely in the intestine, heart, aorta, buccal mass, lips, and foot. However, CARP-immunoreactive cell bodies could only be demonstrated in the intestine and the foot musculature. Thin varicose CARP-immunoreactive fibers were observed over both muscle and gland cells in the different peripheral organs, suggesting a peripheral role of CARP. In vivo CARP injection into the body cavity (10^-3, 10^-4, 10^-5 M) altered the general behavioral state of the animals and induced the relaxation of the musculature of the whole body wall indicating that CARP has a significant role in the regulation of muscle contraction.     

Immunohistochemical localisation of natriuretic peptides in the brains and hearts of the spiny dogfishSqualus acanthias and the Atlantic hagfishMyxine glutinosa

Summary The avidin-biotin peroxidase technique was used to determine the distribution of natriuretic peptides in the hearts and brains of the dogfishSqualus acanthias and the Atlantic hagfishMyxine glutinosa. Three antisera were used: one raised against porcine brain natriuretic peptide which cross-reacts with atrial natriuretic and C-type natriuretic peptides (termed natriuretic peptide-like immunoreactivity); the second raised against porcine brain natriuretic peptide which cross-reacts with C-type natriuretic peptide, but not with atrial natriuretic peptide (termed porcine brain natriuretic peptide-like immunoreactivity); and the third raised against rat atrial natriuretic peptide (termed rat atrial natriuretic peptide-like immunoreactivity). Only natriuretic peptide-like immunoreactivity was observed in the heart ofS. acanthias which was most likely due to the antiserum cross-reacting with C-type natriuretic peptide. No immunoreactivity was found in theM. glutinosa heart. In the brain ofS. acanthias, natriuretic peptide-like immunoreactive fibres were located in many areas of the telencephalon, diencephalon, mesencephalon, rhombencephalon, and spinal cord. Extensive immunoreactivity was observed in the hypothalamo-hypophyseal tract and the neurointermediate lobe of the hypophysis. Natriuretic peptide-like immunoreactive perikarya were found in ventromedial regions of the telencephalon and in the nucleus preopticus. Most perikarya had short, thick processes which extended toward the ventricle. Another group of perikarya was observed in the rhombencephalon. Porcine brain natriuretic peptide-like immunoreactive fibres were observed in the telencephalon, diencephalon, mesencephalon, and rhombencephalon, but perikarya were only present in the preoptic area. In theM. glutinosa brain, natriuretic peptide-like immunoreactive fibres were present in all regions. Immunoreactive perikarya were observed in the pallium, primordium hippocampi, pars ventralis thalami, pars dorsalis thalami, nucleus diffusus hypothalami, nucleus profundus, nucleus tuberculi posterioris, and nucleus ventralis tegmenti. Procine brain natriuretic peptide-like immunoreactive perikarya and fibres had a similar, but less abundant distribution than natriuretic peptide-like immunoreactive structures. Although the chemical structures of natriuretic peptides in the brains of dogfish and hagfish are unknown, these observations show that a component of the natriuretic peptide complement is similar to porcine brain natriuretic peptide or porcine C-type natriuretic peptide. The presence of natriuretic peptides in the brain suggest they could be important neuromodulators and/or neurotransmitters. Furthermore, there appears to be divergence in the structural forms of natriuretic peptides in the hearts and brains of dogfish and hagfish.     

Coexistence of NADPH-diaphorase and vasoactive intestinal polypeptide in the enteric nervous system of the Atlantic cod (Gadus morhua) and the spiny dogfish (Squalus acanthias)

The distribution of NADPH (nicotinamide adenine dinucleotide phosphate)-diaphorase in nerve cells in the gastrointestinal tract has been investigated and compared in three fish species representing different evolutionary branches. In mammals, NADPH-diphorase is identical to nitric oxide synthase (NOS) and can, in the presence of NADPH, reduce the dye nitroblue tetrazolium, resulting in a blue product. Using this method, we have found numerous NADPH-diaphorase-containing nerve cells in the myenteric plexus of the Atlantic cod (Gadus morhua) and the spiny dogfish (Squalus acanthias) but none in the hagfish (Myxine glutinosa). In the cod, nerve fibres were sparsely stained, whereas in the dogfish, they formed a dense pattern of fibre bundles. Double-staining for NADPH-diaphorase and the neuropolypeptides VIP (vasoactive intestinal polypeptide) and PACAP (pituitary adenylate cyclase activating peptide) revealed three separate populations designated VIP/NADPH, VIP/- and NADPH/-. The majority but not all of the NADPH-diaphorase-positive cells also showed VIP or PACAP immunoreactivity and vice versa. The presence of NADPH-diaphorase in neurons and the distribution of these neurons in the gastrointestinal tract of the two species indicate a physiological role for nitric oxide in the control of gut motility.     

Neuronal background of activation of estivated snails,with special attention to the monoaminergic system: a biochemical,physiological, and neuroanatomical study

Osmotic stimulation activates both estivated and inactivated specimens of Helix pomatia and increases their central arousal. High-pressure liquid chromatography has shown that, during activation, the level of both serotonin and dopamine decreases in the central nervous system (CNS) but increases in the foot and heart, organs that are involved in the eversion of the body. In isolated CNS from activated animals, the firing frequency of the heart-modulator serotonergic (RPas) neurons is significantly higher than that in the CNS of estivated or inactivated animals. These neurons innervate both the heart and the anterior aorta. In semi-intact preparations, distilled water (an osmotic stimulus) applied to the mantle collar increases their firing frequency, whereas tactile stimulation evokes their inhibition. Extracellularly applied monoamines mimic the effect of peripheral stimuli: serotonin (0.1–10 μM) increases the activity of the RPas neurons, whereas dopamine (0.1–10 μM) inhibits their activity. Tyrosine-hydroxylase immunocytochemistry and retrograde neurobiotin tracing have revealed similar bipolar receptor cells in the mantle collar and tail, organs that are exposed to environmental stimuli in estivated animals. Serotonin immunocytochemistry carried out on the same tissues does not visualize receptor cells but labels a dense network of fibers that appear to innervate neurobiotin-labeled receptor cells. The combination of neurobiotin-labeling of RPas neurons and immunolabeling suggests that RPas neurons receive direct dopaminergic inputs from receptor cells and serotonergic inputs from central serotonergic neurons, indicating that central serotonergic neurons are interconnected. Thus, the RPas neurons may belong to neuronal elements of the arousal system. This work was supported by Hungarian OTKA grants T037389, T046580, T037505, and K63451.